Recent years have seen increasing promises of using antibodies as diagnostic and therapeutic agents for various disorders and diseases. Many research and clinical applications require large quantities of functional antibodies, thus calling for large scale, economic production systems to be employed. Particularly useful is the recombinant production of antibodies using a variety of expression hosts, ranging from prokaryotes such as E. coli or B. subtilis, to yeast, plants, insect cells and mammalian cells. Kipriyanov and Little (1999) Mol. Biotech. 12:173-201.
Compared to other antibody production systems, bacteria, particularly E. coli, provides many unique advantages. The raw materials used (i.e. bacterial cells) are inexpensive and easy to grow, therefore reducing the cost of products. Shorter generation time and ease of scaling up make bacterial fermentation a more practical means for large-scale protein production. The genomic structure and biological activity of many bacterial species, such as E. coli, have been well-studied and a wide range of expression vectors are available, making expression of a desirable antibody more convenient. Compared with eukaryotes, fewer steps are involved in the production process, including the manipulation of recombinant genes, stable transformation of multiple copies into the host, expression induction and characterization of the products. Pluckthun and Pack Immunotech 3:83-105 (1997). In addition, E. coli permits a unique access to random approaches. Because of the unparalleled efficiency for transformation by plasmids or transfection by phages, E. coli systems can be used for phage library construction of many types of antibody variants, which is particularly important in functional genomic studies.
Currently, bacterial systems are used to produce antibody fragments. Like any other heterologous proteins, antibody fragments can be produced in E. coli either through refolding of inclusion bodies expressed in the cytoplasm, or through expression followed by secretion to the bacterial periplasm. The choice between secretion and refolding is generally guided by several considerations. Secretion is generally the faster and more commonly used strategy.
Opper et al., U.S. Pat. No. 6,008,023, describe an E. coli cytoplasmic expression system, wherein antibody fragments (e.g., Fabs) are fused with an enzyme for use in targeted tumor therapy. Zemel-Dreasen et al. Gene 27:315-322 (1984) report the secretion and processing of an antibody light chain in E. coli. Lo et al's PCT publication, WO 93/07896, reports the E. coli production of a tetrameric antibody lacking the CH2 region in its heavy chain. The genes encoding the light chain and the CH2-deleted heavy chain were constructed into the same expression vector, under the control of one single promoter. The authors acknowledged that the expression system was not optimized and the expression level was moderate. A similar polycistronic system, wherein two expression units (i.e., cistrons) were under the control of one promoter, was used by Carter et al. in U.S. Pat. No. 5,648,237, for producing antibody fragments in E. coli. 
In contrast to the widespread uses of bacterial systems for expressing antibody fragments, there have been few attempts to express and recover at high yield functional intact antibodies in E. coli. Because of the complex feature and large size of an intact antibody, it is often difficult to achieve proper folding and assembly of the expressed light and heavy chain polypeptides, resulting in poor yield of reconstituted tetrameric antibody. Furthermore, since antibodies made in prokaryotes are not glycosylated, thus lacking the effector functions, the art has suggested that E. coli would not be a useful system for making intact antibodies. Pluckthun and Pack (1997) Immunotech 3:83-105; Kipriyanov and Little Mol. Biotech. 12:173-201 (1999); Pluckthun et al. (1996) in ANTIBODY ENGINEERING: A PRACTICAL APPROACH, pp 203-252 (Oxford Press); Pluckthun (1994) in HANDBOOK OF EXP. PHARMCOL vol 3: The Pharmcol. of Monoclonal Antibodies, pp 269-315 (ed. M. Rosenberg and G. P. Moore; Springer-Verlag, Berlin).
Recent developments in research and clinical studies suggest that in many instances, intact antibodies are preferred over antibody fragments. An intact antibody containing the Fc region tends to be more resistant against degradation and clearance in vivo, thereby having longer biological half life in circulation. This feature is particularly desirable where the antibody is used as a therapeutic agent for diseases requiring sustained therapies.
Furthermore, in many instances, intact antibodies deficient in effector functions are more desirable for therapeutic uses. Friend et al., Transplantation 68: 1632-1637 (1999) describe toxic effects, such as severe cytokine release syndromes, of glycosylated CD3 monoclonal antibodies when used in humans for the treatment of acute rejection episodes of organ allografts. The CD3 antibodies cause T-cell activation and cytokine release by cross-linking the T cell receptor complex as a result of FcR binding. U.S. Pat. No. 5,585,097 describe making aglycosylated CD3 antibodies by mutating certain glycosylation site residues of native CD3 antibodies. Armour et al., Eur. J. Immunol. 29:2613-2624 (1999) describe the use of non-destructive antibodies (i.e., lacking the effector functions) specific for HPA-1a-positive platelets in therapeutic applications where depletion of cells bearing the target antigen (i.e., the platelet cells) is undesirable. Thompson, et al., J. Immunol Meth 227:17-29 (1999) show that effector functions of a fully human antibody against TGFβ2 are not necessary for use in therapy of fibrotic diseases mediated by TGFβ2. Reddy, et al., J. Immunol. 164:1925-1933 (2000) describe liability of strong antibody-Fcγ receptor binding in treating autoimmune diseases; Isaacs, et al., Clin. Exp. Immunol. 106:427-433(1996) suggest that if a pure blocking effect is required in vivo, an aglycosylated monoclonal antibody variant or a mutant engineered to prevent Fc receptor binding may be better choices.
Currently, attempts to eliminate or reduce effector functions of an antibody focus on either using IgG4 isotype, which is thought to be unable to deplete target cells, or making Fc variants, wherein residues in the Fc region critical for effector function(s) are mutated. See, for example, U.S. Pat. No. 5,585,097. However, both of these approaches have limitations. For example, the IgG4 isotype has been shown to retain some level of effector functions, as described by Isaacs, et al. (1996) supra, and Thompson, et al. (1999), supra. Reddy et al. (2000), supra, also report that further alterations of an IgG4 mAb against CD4 were required to eliminate Fc effector functions. Fc mutants may elicit undesirable immune response because of the residue changes in the primary sequence.